Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site.

Nataliya Bohdan,Irene Martínez-Martínez,Vicente Vicente,Sonia Águila,Raúl Teruel-Montoya,Javier Corral,Salvador Espín,Nikos K Karamanos

doi:10.1371/journal.pone.0157834

Abstract

Heparanase is an endoglycosidase that participates in morphogenesis, tissue repair, heparan sulphates turnover and immune response processes. It is over-expressed in tumor cells favoring the metastasis as it penetrates the endothelial layer that lines blood vessels and facilitates the metastasis by degradation of heparan sulphate proteoglycans of the extracellular matrix. Heparanase may also affect the hemostatic system in a non-enzymatic manner, up-regulating the expression of tissue factor, which is the initiator of blood coagulation, and dissociating tissue factor pathway inhibitor on the cell surface membrane of endothelial and tumor cells, thus resulting in a procoagulant state. Trying to check the effect of heparanase on heparin, a highly sulphated glycosaminoglycan, when it activates antithrombin, our results demonstrated that heparanase, but not proheparanase, interacted directly with antithrombin in a non-covalent manner. This interaction resulted in the activation of antithrombin, which is the most important endogenous anticoagulant. This activation mainly accelerated FXa inhibition, supporting an allosteric activation effect. Heparanase bound to the heparin binding site of antithrombin as the activation of Pro41Leu, Arg47Cys, Lys114Ala and Lys125Alaantithrombin mutants was impaired when it was compared to wild type antithrombin. Intrinsic fluorescence analysis showed that heparanase induced an activating conformational change in antithrombin similar to that induced by heparin and with a KD of 18.81 pM. In conclusion, under physiological pH and low levels of tissue factor, heparanase may exert a non-enzymatic function interacting and activating the inhibitory function of antithrombin.

Highlights

Heparanase is an endoglycosidase able to cleave heparan sulphate side chains at a limited number of sites, yielding heparan sulphate fragments of still appreciable size (~5–7 kDa) [1,2,3]
Incubation of antithrombin with heparanase in the absence of heparin provoked the activation of antithrombin in its inhibitory function of FXa (Fig 1A), and slightly increased the inhibitory effect on FIIa (Fig 1B), indicating that there is an allosteric activation of antithrombin
Heparanase did not impair the activation of antithrombin by Low molecular weight heparin (LMWH) or unfractionated heparin (UFH)

Summary

Introduction

Heparanase is an endoglycosidase able to cleave heparan sulphate side chains at a limited number of sites, yielding heparan sulphate fragments of still appreciable size (~5–7 kDa) [1,2,3]. Heparanase is secreted by platelets after degranulation, leukocytes or endothelial cells and it has multiple roles. It can aid cell invasion by degrading heparan sulphate proteoglycans and can release growth factors bound to heparan-sulphate that initiate angiogenesis, such as VEGF, or activate tissue repair by releasing FGF. Heparan sulphate-disaccharides liberated by heparanase inhibit TNF production by macrophages with direct consequences as a negative regulator of inflammation [4]. Up-regulated expression of heparanase has been noted in essentially all human tumors examined, as well as in inflammation, wound healing, and diabetic nephropathy [5,6,7]

Methods

Results

Conclusion